Esters of acyl carnitines with long-chain aliphatic alcohols and pharmaceutical compositions containing same having antibacterial activity

ABSTRACT

Esters of acyl L-carnitines of general formula (I) ##STR1## wherein R is a straight or branched acyl group having 2 to 16 carbon atoms, in particular isobutyryl and isovaleryl; 
     n is an integer comprised between 7 and 15, particularly 10; and, 
     X- is the anion of a pharmacologically acceptable acid are endowed with potent antibacterial activity. 
     Pharmaceutical compositions comprising an ester of formula (I) can be utilized in human therapy and in the veterinary field.

This application is a Continuation of application Ser. No. 08/000,710,filed on Jan. 5, 1993, now abandoned.

The present invention relates to esters of acyl L-carnitines withlong-chain aliphatic alcohols, of general formula (I) ##STR2## wherein Ris a straight or branched saturated carboxylic acyl group having from 2to 16, preferably from 4 to 12, carbon atoms, in particular isobutyryland isovaleryl;

n is an integer comprised between 7 and 15, that is from 7 to 15,particularly 10, and

X- is the anion of a pharmacologically acceptable acid.

Among the esters of formula (I) isovaleryl L-carnitine undecil ester andisobutyryl L-carnitine undecil ester are particularly preferred.

The esters of formula (I) are endowed with a potent antibacterialactivity against Gram⁺ and Gram⁻ bacteria, in particular againstbacteria of the genera Campylobacter and Helicobacter, which arerespectively the aetiologic agents of intestinal infections in humansand of intestinal and genital infections in animals (Campylobacterfetus, jejuni, coli) or responsible of B-type gastritis and duodenalulcer in humans (Helicobacter pylori).

Therefore, the present invention also relates to orally or parenterallyadministrable pharmaceutical compositions useful in human therapy forthe treatment of intestinal infections, B-type gastritis and duodenalulcer and also relates to pharmaceutical compositions useful for thetreatment of intestinal and genital infections in animals.

The genus Campylobacter which encompasses microaerophilic Gram negativebacteria has recently drawn the attention of microbiologists because itsrole as aetiologic agent has been ascertained in a number of human andanimal pathologies of growing impact and increasingly difficulttreatment.

Numerous strains appear to exhibit intrinsic resistance to manyantibiotics, probably due to the inability of the drug (Bacitracin,Novobiocin, Rifampicin, Streptogramin B, Trimethoprim, Vancomycin,Cephalotyn) to penetrate the bacteria cells.

Other types of resistance may be correlated to acquired resistance ofboth plasmid type (Tetracycline, Kanamycin, Chloramphenicol) withsubsequent ribosomial protection or 3'-aminoglycoside phosphotransferaseproduction, and chromosomial type (Streptomycin, Spectinomycin,Erythromycin, Ampicillin, Nalidixic acid) brought about by theproduction of 6-aminoglycoside nucleotransferase or β-lattamase.

Recently, on the basis of recent advances in their culture,biochemistry, morphology and susceptibility to antibiotics, the genusHelicobacter comprising the species pylori and mustelae has beendifferentiated from the genus Campylobacter.

The species fetus, jejuni and coli of the genus Campylobacter were shownto be responsible for intestinal infections in humans and for intestinaland genital infections in animals.

On the other hand, the genus Helicobacter has been linked to B-typegastritis in humans, since it was repeatedly found in the inflammatorytissue of the gastric mucosa of patients with gastritis (Marshall B. J.WARREN J. R., 1984, Unidentified curved bacilli in the stomach ofpatients with gastritis and peptic ulceration, LANCET. 1:1311-1313).

A very close relationship has been recently hypothesized among theinfection by H. pylori, type-B gastritis, duodenal ulcer and gastriccarcinoma (BLASER M. J. 1990, "Helicobacter pylori" and the Pathogenesisof Gastroduodenal Inflammation. The J. of Infect. Dis. 161: 626-633).

A strong interest for developing new compounds endowed withantibacterial activity against the genus Helicobacter and,subordinately, against the genus Campylobacter can be accounted for onthe grounds of the foregoing studies and the high frequency of relapsesnoticed in the last years.

The esters of formula (I) may be prepared following two distinctsynthesis processes. The first process (illustrated in the synthesisscheme 1) comprises the steps consisting of:

(a) halogenating an acyl L-carnitine with a halogenating agent, such asthionyl chloride and oxalyl chloride (molar ratio comprised between 1:1and 1:4) in an anhydrous organic inert solvent such as acetonitrile ormethylene chloride at a temperature comprised between 0° C. and 30° C.for 1-4 hours, concentrating the raw reaction product and using it inthe following step;

(b) dissolving the acid chloride of step (a) in an anhydrous organicinert solvent such as acetonitrile or methylene chloride and adding thealcohol diluted in the same solvent at a ratio comprised between 1:1 and1:2, at a temperatures comprised between 0° C. and 30° C. for 2-10hours, concentrating the solution and, if needed, purifying the compoundby chromatography on silica gel; and

(c) eluting the product dissolved in water or in an organic solvent on astrongly basic ion exchange resin such as Amberlite IRA 402 or on aweakly basic ion exchange resin such as Amberlist A 21, activated withthe desired HX acid and isolating the end product by lyophilization orconcentration.

The second process (illustrated in the synthesis scheme 2) comprise thesteps consisting of:

(a') reacting carnitine or an acyl carnitine inner salt with therelevant alkyl halogenide (preferably bromide or iodide) in an organicanhydrous inert solvent at a temperature comprised between 30° C. and60° C. for 8-24 hours and then isolating the resulting compound byconcentration;

(b') acylating the ester obtained in step (a') with the desired acidchloride by known techniques, in case the starting compound in step (a')is carnitine;

(c') eluting an aqueous or alcoholic solution of the compound of step(a') or (b') on an ion exchange resin, such as Amberlite IRA 402 orAmberlist A 21 activated with the desired HX acid.

The anion X⁻ of the pharmacologically acceptable acid is preferablyselected from chloride; bromide; iodide; aspartate, particularly acidasparatate; citrate, particularly acid citrate; tartrate; phosphate,particularly acid phosphate; fumarate, particularly acid fumarate;glycerophosphate; glucosephosphate; lactate; maleate, particularly acidmaleate; orotate; oxalate, particularly acid oxalate; sulphate,particularly acid sulphate; trichloroacetate; trifluoroacetate andmethansulphonate. ##STR3##

EXAMPLE 1 Preparation of Isovaleryl-L-Carnitine Undecyl Ester Chloride(ST 722)

Step A: Preparation of Isovaleryl-L-Carnitine Chloride Acid Chloride

Isovaleryl-L-carnitine chloride (30 g; 0.106 moles) was suspended in 100ml anhydrous CH₂ Cl₂.

The mixture was cooled at 0° C. and oxalyl chloride (13 ml; 0.15 moles)diluted in 15 ml anhydrous CH₂ Cl₂ was slowly added under stirring.

After 30 minutes at room temperature, a further amount of oxalylchloride (19 ml; 0.21 moles) diluted in 10 ml anhydrous CH₂ Cl₂ wasadded.

The resulting solution was kept under stirring for 2 hours at roomtemperature, then concentrated under vacuum.

The residue thus obtained was washed twice with anhydrous CH₂ Cl₂ andconcentrated under vacuum.

The raw product thus obtained was used as such in the next reaction.

Step B: Preparation of Isovaleryl-L-Carnitine Undecyl Ester Chloride (ST722)

The acid chloride previously prepared (0.106 moles) was dissolved inanhydrous CH₂ Cl₂ (40 ml).

The solution was cooled at 0° C. and undecylic acid (35 ml; 0.168 moles)diluted in 35 ml CH₂ Cl₂ was added in a nitrogen atmosphere.

The solution was kept under stirring at room temperature for 2 hours andthen concentrated under vacuum until an oily residue was obtained.

The raw reaction mixture was chromatographed on a silica gel columnbuffered with 2% Na₂ HPO₄, eluting with CH₂ Cl₂ till complete elution ofundecylic alcohol and then with CH₂ Cl₂ --MeOH 9:1 till complete elutionof the compound.

The pooled fractions were concentrated and gave 28 g of the titlecompound; Yield 60%.

[α]_(D) ²⁵ =-10.5 (c=1% H₂ O)),

    ______________________________________                                        Elementary analysis for C.sub.23 H.sub.46 ClNO.sub.4                                         C %  H %     Cl %    N %                                       ______________________________________                                        Calculated (anhydrous)                                                                         63.35  10.63   8.13  3.21                                    Found            60.87  0.88    8.14  3.29                                    ______________________________________                                    

H₂ O 2.4%, HPLC Column: Spherisorb Cl 5 μm, t.: 50° C., Eluant: CH₃OH/KH₂ PO₄ 50 mM (65:35), Flow rate: 1 ml/min., Retention time: 14.82min. ##STR4##

EXAMPLE 2 Preparation of Isobutyryl-L-Carnitine Undecyl Ester Chloride(ST 712)

The compound was prepared as described in example 1, substitutingisobutyril L-carnitine chloride. for isovaleryl L-carnitine chloride.Yield 55%.

[α]_(D) ²⁵ =-15.8 (c=1% H₂ O),

    ______________________________________                                        Elementary analysis for C.sub.22 H.sub.44 O.sub.4 NCl                                        C %  H %     Cl % N  %                                         ______________________________________                                        Calculated (anhydrous)                                                                         62.61  10.51   3.32  8.40                                    Found            61.77  10.67   3.29  8.17                                    ______________________________________                                    

H₂ O 0.8%, HPLC Column: Spherisorb Cl (4.6 mm), eluant CH₃ OH--KH₂ PO₄50 mM 60-40, Flow rate: 1 ml/min, Retention time: 14,75 min. ##STR5##

EXAMPLES 3-19

The compounds of Examples 3-19 were prepared following the procedures ofthe previous examples, as apparent to any average expert in organicsynthesis. The physico-chemical characteristics of the compounds aresummarized in the following table.

    __________________________________________________________________________    Ex               [α]25                                                                             E.A. found          m.p.  HPLC                     code R     n X.sup.-                                                                           D         C   H   N   Cl  H.sub.2 O                                                                         °C.                                                                          Rt                       __________________________________________________________________________                                                         min                      3                          C.sub.26 H.sub.52 NO.sub.4 Cl                      ST 1000                                                                            octanoyl                                                                            10                                                                              Cl.sup.-                                                                          -10,7     62,84%                                                                            10,7%                                                                             3,03%                                                                             6,96%                                                                             3,2%                                                                              103-105                                                                             10,28.sup.a                               (c = 1% CHCl.sub.3)                                          4                          C.sub.29 H.sub.58 NO.sub.4 Cl                      ST 982                                                                             undecanoyl                                                                          10                                                                              Cl.sup.-                                                                          -14       66,65%                                                                            11,72%                                                                            2,70%                                                                             6,93%                                                                             0,4%                                                                              133-135                                                                             16,32.sup.b                               (c = 0,5% MetOH)                                             5                          C.sub.34 H.sub.68 NO.sub.4 Cl                      ST 983                                                                             palmitoyl                                                                           10                                                                              Cl.sup.-                                                                          -14       69,42%                                                                            11,84%                                                                            2,36%                                                                             5,96%                                                                             0,8%                                                                              158- 159                                                                            10,5.sup.c                                (c = 1% MetOH)                                               6                          C.sub.24 H.sub.48 NO.sub.4 Cl                      ST 1034                                                                            isocaproyl                                                                          10                                                                              Cl.sup.-                                                                           -13,12   61,28%                                                                            11,10%                                                                            3,12%                                                                             9,01%                                                                             1,9%                                                                              oil/not                                                                              8,45.sup.d                               (c = 0,8% H.sub.2 O)          determined                     7                          C.sub.25 H.sub.49 NO.sub.4 Cl                      ST 1036                                                                            heptanoyl                                                                           10                                                                              Cl.sup.-                                                                          -12,1     64,35%                                                                            12,55%                                                                            3,09%                                                                             6,68%                                                                             1,3%                                                                              not    9,35.sup.d                               (c = 1% H.sub.2 O)            determined                     8                          C.sub.27 H.sub.54 NO.sub.4 Cl                      ST 1050                                                                            heptanoyl                                                                           12                                                                              Cl.sup.-                                                                          -10,3     65,26%                                                                            11,62%                                                                            2,87%                                                                             6,70%                                                                             0,3%                                                                              dec.   9,13.sup.e                               (c = 0,7% CHCl.sub.3)         150-160                        9                          C.sub.27 H.sub.54 NO.sub. 4 Cl                     ST 1051                                                                            2-methyl                                                                            12                                                                              Cl.sup.-                                                                           -8,8     65,06%                                                                            11,32%                                                                            2,91%                                                                             6,93%                                                                             0,4%                                                                              not   28,03.sup.b                   hexanoyl    (c = 1% CHCl.sub.3)           determined                     10                         C.sub.25 H.sub.50 NO.sub.4 Cl                      ST 1033                                                                            isovaleryl                                                                          12                                                                              Cl.sup.-                                                                          -11,8     63,73%                                                                            12,50%                                                                            3,17%                                                                             7,03%                                                                             1,2%                                                                              dec.   9,39.sup.d                               (c = 1% H.sub.2 O)            150                            11                         C.sub.26 H.sub.52 NO.sub.4 Cl                      ST 1052                                                                            hexanoyl                                                                            12                                                                              Cl.sup.-                                                                          -10,7     65,01%                                                                            11,87%                                                                            2,93%                                                                             7,14%                                                                             1,2%                                                                              dec.  14,86.sup.c                               (c = 1% CHCl.sub.3)           120-130                        12                         C.sub.28 H.sub.56 NO.sub.4 Cl                      ST 1053                                                                            octanoyl                                                                            12                                                                              Cl.sup.-                                                                           -9,8     66,46%                                                                            11,93%                                                                            2,71%                                                                             6,93%                                                                             0,7%                                                                              dec.  14,71.sup.c                               (c = 1% CHCl.sub.3)           150-160                        13                         C.sub.24 H.sub.48 NO.sub.4 Cl                      ST 1037                                                                            isovaleryl                                                                          11                                                                              Cl.sup.-                                                                          -12,2     63,46%                                                                            12,26%                                                                            3,15%                                                                             7,81%                                                                             1,0%                                                                              dec.  12,65.sup.a                               (c = 1% H.sub.2 O)            150-160                        14                         C.sub.23 H.sub.46 NO.sub.4 Cl                      ST 1038                                                                            isobutiryl                                                                          11                                                                              Cl.sup.-                                                                          -14,5     62,90%                                                                            11,47%                                                                            3,27%                                                                             7,86%                                                                             0,4%                                                                              dec.  14,0.sup.a                                (c = 1% H.sub.2 O)            150-155                        15                         C.sub.26 H.sub.52 NO.sub.4 Cl                      ST 1060                                                                            heptanoyl                                                                           11                                                                              Cl.sup.-                                                                          -12,7     67,00%                                                                            12,12%                                                                            2,41%                                                                             6,60%                                                                             0,6%                                                                              not   10,47.sup.f                               (c = 1% MetOH)                determined                     16                         C.sub.28 H.sub.56 NO.sub.4 Cl                      ST 1001                                                                            isovaleryl                                                                          15                                                                              Cl.sup.-                                                                          -12,6     65,30%                                                                            11,11%                                                                            2,68%                                                                             7,57%                                                                             0,5%                                                                              not   12,12.sup.a                               (c = 0,5% H.sub.2 O)          determined                     17                         C.sub.27 H.sub.51 NO.sub.10                        ST 1018                                                                            isovaleryl                                                                          10                                                                              tartrate                                                                           -1,9     56,87%                                                                             9,78%                                                                            2,44%   4%  not   13,74.sup.a                           acid                                                                              (c = 1% H.sub.2 O)            determined                     18                         C.sub.27 H.sub.49 NO.sub.8                         ST 1017                                                                            isovaleryl                                                                          10                                                                              fuma                                                                              -13,3%    62,23%                                                                             9,90%                                                                            2,54%   0,7%                                                                              dec.  13,99.sup.a                           rate                                                                              (c = 1% H.sub.2 O)            120                                         acid                                                             19                         C.sub.20 H.sub.40 ClNO.sub.4                       ST 1032                                                                            isovaleryl                                                                           8                                                                              Cl.sup.-                                                                          -17,4%    59,93%                                                                            10,25%                                                                            3,49%                                                                             8,84%                                                                             1,7%                                                                              not    5,75.sup.b                               (c = 1% H.sub.2 O)            determined                     __________________________________________________________________________                                  Ex                                                                            code NMR δ                                __________________________________________________________________________                                  3    5,7(1H, m, CHO); 4,3-4,0(4H, m,                                               CH.sub.2 N.sup.+ ;                                                       ST 1000                                                                            OCH.sub.2); 3,5(9H, s,                                                        (CH.sub.3).sub.3 N.sup.+); 2,8 (2H,                                           m,                                                                            CH.sub.2 COO); 2,3(2H, t,                                                     OCOCH.sub.2); 1,8(4H, m,                                                      2 CH.sub.2); 1,6(4H, m; 2 CH.sub.2);                                          1,3(20H, broad,                                                               10 CH.sub.2); 0,9(6H, 2t, 2 CH.sub.3).                                        CDCl.sub.3                                                               4    5,6(1H, m, CHO); 4,1(2H, t,                                                   OCH.sub.2); 3,9-3,7                                                      ST 982                                                                             (2H, m, CH.sub.2 N.sup.+); 3,3(9H, s,                                         (CH.sub.3).sub.3 N.sup.+); 2,8                                                (2H, dd, CH.sub.2 COO); 2,4(2H, t,                                            OCOCH.sub.2);                                                                 1,6(4H, m, 2CH.sub.2); 1,3(30H,                                               broad,                                                                        15CH.sub.2); 0,9(6H, t, 2CH.sub.3).                                           CD.sub.3 OD                                                              5    5,6(1H, m, CHO); 4,1(2H, t,                                                   OCH.sub.2); 3,9-3,7                                                      ST 983                                                                             (2H, m, CH.sub.2 N.sup.+); 3,2(9H, s,                                         (CH.sub.3).sub.3 N.sup.+);                                                    2,8(2H, m, CH.sub.2 COO); 2,4(2H, t,                                          OCOCH.sub.2);                                                                 1,6(4H, m, 2CH.sub.2); 1,3(40H, broad,                                        20CH.sub.2);                                                                  0,9(6H, t, 2CH.sub.3). CD.sub.3 OD                                       6    5,7(1H, m, CHO); 4,1(2H, t,                                                   OCH.sub.2); 4,0-                                                         ST 1034                                                                            3,7(2H, m, CH.sub.2 N.sup.+); 3,2(9H,                                         s, (CH.sub.3).sub.3 N.sup.+);                                                 3,0-2,7(2H, m, CH.sub.2 COO);                                                 2,6-2,3(2H, m,                                                                OCOCH.sub.2); 1,7-1,4(5H, m,                                                  2CH.sub.2,H); 1,3                                                             (16H, broad, 8CH.sub.2); 0,9 (6H, d,                                          (CH.sub.3).sub.2);                                                            0,8(3H, t, CH.sub.3). D.sub.2 O                                          7    5,7(1H, m, CHO); 4.1(2H, t,                                                   OCH.sub.2); 4,0-3,7                                                      ST 1036                                                                            (2H, m, CH.sub.2 N.sup.+); 3,2(9H, s,                                         (CH.sub.3).sub.3 N.sup.+);                                                    3,0-2,7(2H, m, CH.sub.2 COO);                                                 2,5-2,3(2H, m,                                                                COCH.sub.2); 1.6(4H, m, 2CH.sub.2);                                           1,3(22H, m,                                                                   11CH.sub.2); 0,9-0,8(6H, 2t,                                                  2CH.sub.3). D.sub.2 O                                                    8    5,7(1H, m, CHO); 4,3-4,0(4H, m,                                               CH.sub.2 N.sup.+ ;                                                       ST 1050                                                                            OCH.sub.2); 3,5(9H, s,                                                        (CH.sub.3).sub.3 N.sup.+); 2,8(2H, m,                                         CH.sub.2 COO); 2,3(2H, m,                                                     OCOCH.sub.2); 1,6(4H,                                                         m, 2CH.sub.2); 1,3(26H, m,                                                    13CH.sub.2); 0,9(6H, 2t,                                                      2CH.sub.3). CDCl.sub.3                                                   9    5,7(1H, m, CHO); 4,3-4,0(4H, m,                                               CH.sub.2 N.sup.+ ;                                                       ST 1051                                                                            OCH.sub.2); 3,5(9H, s,                                                        (CH.sub.3).sub.3 N.sup.+); 2,8(2H, m,                                         CH.sub.2 COO); 2,4(1H, m, CH); 1,6(2H,                                        m,                                                                            CH.sub.2); 1,3(26H, m, 13 CH.sub.2);                                          1,1(3H, m,                                                                    CHCH.sub.3); 0,9(6H, 2t, 2CH.sub.3).                                          CDCl.sub.3                                                               10   5,7(1H, m, CHO); 4,1(2H, m,                                                   OCH.sub.2); 4,0-3,7                                                      ST 1033                                                                            (2H, m, CH.sub.2 N.sup.+); 3,2(9H, s,                                         (CH.sub.3).sub.3 N.sup.+); 3,0-                                               2,7(2H, m, CH.sub.2 COO); 2,3(2H, m,                                          OCOCH.sub.2);                                                                 2,1(1H, m, CH.sub.2 CH); 1,6(2H, m,                                           CH.sub.2); 1,3                                                                (20H, broad, 11 CH.sub.2); 0,9(6H,                                            dd,                                                                           CH(CH.sub.3)2); 0,8(3H, t, CH.sub.3).                                         D.sub.2 O                                                                11   5,7(1H, m, CHO); 4,3-4,0(4H, m,                                          ST 1052                                                                            CH.sub.2 N.sup.+ ; OCH.sub.2); 3,5(9H,                                        s, (CH.sub.3 ).sub.3 N.sup.+); 2,9-                                           2,7(2H, m, CH.sub.2 COO); 2,3(2H, t,                                          OCOCH.sub.2);                                                                 1,6(4H, m, 2CH.sub.2); 1,3(24H, broad,                                        12 CH.sub.2);                                                                 0,9(6H, m, 2CH.sub.3). CDCl.sub.3                                        12   5,7(1H, m, CHO); 4,3-4,0(4H, m,                                          ST 1053                                                                            CH.sub.2 N.sup.+ ; OCH.sub.2); 3,5(9H,                                        s, (CH.sub.3).sub.3 N.sup.+); 2,9-                                            2,7(2H, m, CH.sub.2 COO); 2,3(2H, m,                                          OCOCH.sub.2);                                                                 1,6(4H, m, 2CH.sub.2); 1,3(28H, broad,                                        14 CH.sub.2);                                                                 0,9(6H, m, 2CH.sub.3). CDCl.sub.3                                        13   5,7(1H, m, CHO); 4,4-4,0(4H, m,                                          ST 1037                                                                            N.sup.+ CH.sub.2 ; OCH.sub.2) 3,5(9H,                                         s, N.sup.+ (CH.sub.3).sub.3);                                                 2,8(2H, m, CH.sub.2 COO); 2,2(2H, m,                                          OCOCH.sub.2);                                                                 2,0(1H, m, CH(CH.sub.3).sub.2);                                               1,6(2H, m, CH.sub.2);                                                         1,2(18H, broad, 9(CH.sub.2);                                                  0,9-0,8(9H, d+t,                                                              CH.sub.3 ; (CH.sub.3).sub.2).                                                 CDCl.sub.3                                                               14   5,7(1H, m, CHO); 4,4-4,0(4H, m,                                          ST 1038                                                                            N.sup.+ CH.sub.2 ; OCH.sub.2); 3,5(9H,                                        s, N.sup.+ (CH.sub.3).sub.3 );                                                2,9-2,7(2H, m, CH.sub.2 COO);                                                 2,6-2,5(1H, m,                                                                CH(CH.sub.3).sub.2); 1,6(2H, m,                                               CH.sub.2); 1,3(18H,                                                           broad, 9CH.sub.2); 1,1(6H, d,                                                 CH(CH.sub.3).sub.2); 0,8                                                      (3H, t, CH3). CDCl3                                                      15   5,7(1H, m, CHO); 4,4-4,0(4H, m,                                          ST 1060                                                                            N.sup.+ CH.sub.2 ; OCH.sub.2); 3,5(9H,                                        s, N.sup.+ (CH.sub.3).sub.3);                                                 2,8(2H, m, CH.sub.2 COO); 2,4(2H, t,                                          COOCH.sub.2)                                                                  1,6(2H, m, CH.sub.2); 1,3(26H, broad,                                         13 CH.sub.2);                                                                 0,9(6H, 2t, 2CH.sub.3)                                                   16   5,7(1H, m, CHO); 4,4-4,0(4H, m,                                          ST 1001                                                                            N.sup.+ CH.sub.2 ; OCH.sub.2); 3,5(9H,                                        s, N.sup.+ (CH.sub.3).sub.3);                                                 2,8(2H, m, CH.sub.2 COO); 2,2(3H, m,                                          CH(CH.sub.3).sub.2); 1,6(2H, m,                                               CH.sub.2); 1,3(26H,                                                           broad, 13 CH.sub.2); 1,0-0.9(9H, d+t,                                         CH.sub.3, CH(CH.sub.3).sub.2).                                                CDCl.sub.3                                                               17   5,7(1H, m, CHO); 4,5(2H, s, 2CHOH);                                           4,1-                                                                     ST 1018                                                                            3,6(4H, m, N.sup.+ CH.sub.2 ;                                                 OCH.sub.2); 3,2(9H, s, N.sup.+                                                (CH.sub.3).sub.3); 3,0-2,7(2H, m,                                             CH.sub.2 COO); 2,4-                                                           2,2(2H, m, OCOCH.sub.2); 2,1-2,0(1H,                                          m,                                                                            CH(CH.sub.3).sub.2); 1,6(2H, m,                                               CH.sub.2); 1,3(16H,                                                           broad, 8CH.sub.2); 0,9(6H, d,                                                 CH(CH.sub.3).sub.2);                                                          0,8(3H, t, CH.sub.3). D.sub.2 O                                          18   6,6(2H, s, CHCH); 5,7(1H, m, CHO);                                       ST 1017                                                                            4,1-3,7(4H, m, N.sup.+ CH.sub.2 ;                                             OCH.sub.2); 3,2(9H, s, N.sup.+                                                (CH.sub.3).sub.3 ; 3,0-2,7(2H, m,                                             CH.sub.2 COO); 2,4-2,2                                                        (2H, m, OCOCH.sub.2); 2,0(1H, m,                                              CH(CH.sub.3).sub.2);                                                          1,6(2H, m, CH.sub.2); 1,3(16H, broad,                                         8CH.sub.2);0,9                                                                (6H, t, CH(CH.sub.3).sub.2); 0,8(3H,                                          t, CH.sub.3). D.sub.2 O                                                  19   5,7(1H, m, CHO); 3,8-4,1(4H, m,                                               N.sup.+ CH.sub.2 ;                                                       ST 1032                                                                            OCH.sub.2); 3,3(9H, s, N.sup.+                                                (CH.sub.3).sub.3); 2,8(2H, m,                                                 CH.sub.2 COO); 2,3(2H, m, COCH.sub.2);                                        2,1(1H, m,                                                                    CH(CH.sub.3).sub.2); 1,6(2H, m,                                               CH.sub.2); 1,3(10H, m,                                                        broad); 1,0-0,9(9H, d+t, CH.sub.3                                             CH(CH.sub.3).sub.2.                                                           D.sub. 2 O                                 __________________________________________________________________________     .sup.a Column: NucleosilSA (5 μ) 1,2 mm, i.d. 4,0 mm T: 40° C.      mobile phase: (NH.sub.4).sub.2 HPO.sub.4 50 mM/CH.sub.3 CN 1:1 pH 4 con       H.sub.3 PO.sub.4 flow: 0,75 ml/min                                            .sup.b Column: SpherisorbC1 (5 μ) 1,2 mm i.d. 4,6 mm T: 50° C.      mobile phase: CH.sub.3 OH/KH.sub.2 PO.sub.4 50 mM 60:40 flow: 0,5 ml/min      .sup.c Column: SpherisorbC1 (5 μ) 1,2 mm i.d. 4,6 mm T: 50° C.      mobile phase: CH.sub.3 OH/KH.sub.2 PO.sub.4 50 mM 70:30 pH 3,9 con H.sub.     PO.sub.4 flow: 0,5 ml/min                                                     .sup.d Column: SpherisorbC1 (5 μ) 1,2 mm i.d. 4,6 mm T: 40° C.      mobile phase: CH.sub.3 OH/KH.sub.2 PO.sub.4 50 mM 65:35 pH 4,5 con H.sub.     PO.sub.4 flow: 0,5 ml/min                                                     .sup.e Column: NucleosilSA (5 μ) 1,2 mm, i.d. 4,0 mm T: 30° C.      mobile phase: (NH.sub.4).sub.2 HPO.sub.4 50 mM/CH.sub.3 CN 65:35 pH 3,5       con H.sub.3 PO.sub.4 flow: 0,75 ml/min                                         .sup.f Column: SpherisorbC1 (5 μ) 1,2 mm i.d. 4,6 mm T: 40° C.     mobile phase: CH.sub.3 OH/KH.sub.2 PO.sub.4 50 mM 65:35 pH 4,5 con H.sub.     PO.sub.4 flow: 1 ml/min                                                  

ASSESSMENT OF BEHAVIOUR AND MORTALITY IN MICE

The assessment of normal behaviour in mice was carried out following S.Irwin's method (Psychopharmacologia, 13, 222 [1968]). This method allowsalterations in some behavioural, neurophysiologic and neurovegetativeparameters to be detected, which are directly observable by theresearcher. The study was conducted using male Crl:(CD-1)(ICR)BR mice(Charles River--Italy) weighing 22-25 g, following oral administrationof the compounds suspended in carboxymethylcellulose (0.5% by weight inH₂ O) to groups of 4 animals/dose.

The animals were continously kept under observation for five hoursfollowing treatment and twice a day in the subsequent five days.Mortality was also observed during the overall test period.

    ______________________________________                                        Assessment of behaviour and mortality in mice.                                Compound  dose     Symptoms      Mortality                                    ______________________________________                                        ST 712    1000     NONE          O/4                                          ST 722    1000     salivation, diarrhoea                                                                       O/4                                          ______________________________________                                    

IMMUNOTOXICOLOGICAL STUDIES

Some immunotoxicological results after oral ST 722 administration inmice are described hereinafter:

Test 1

Evaluation of the "in vitro-ex vivo" effect of repeated oraladministrations of ST 722 on the primary antibody production (Jernetest) in the spleen of SRBC (Sheep Red Blood Cells) immunized mice.

Experimental Procedure

Male B₆ D₂ F₁ mice (C. River) aged 8 weeks (6 animals each group) wereutilized.

The substance (ST 722) was administered per os at the dose of 100mg/kg/day from day -2 through day +2 (immunization at day 0).

The animals were immunized by intraperitoneal route with a concentrationof 1.0×10⁸ cells/mouse in 0.2 ml of sterile saline.

Five days later, spleens to be submitted to teasing process were removedfrom the animals sacrificed by cervical dislocation.

After standardization at 1.0×10⁷ cells/ml, the splenocytes (0.1 ml) weremixed with warm agar-Hank's (2 ml) and 10% SRBC in PBS (0.2 ml) andseeded in Petri dishes (samples tested in triplicates) and incubated at37° C. for 60 min.

After addition of complement (2 ml of guinea pig serum diluted 1:10 inTris buffer), samples were further incubated at 37° C. for 30 min.

To block the haemolysis reaction, the Petri dishes were cooled at 4° C.and the haemolysis plaques were counted.

The antibody response to SRBC is expressed as number of plaque formingcells (PFC) per 1.0×10⁶ splenocytes as well as per spleen.

Results

The results indicated that the repeated (5 consecutive days) oraladministrations of ST 722 did not cause a statistically significantmodification in PFC number after the antigenic challenge (Table 1).These data exclude the existence of an immunotoxic effect on theantibody-producer B lymphocytes.

The weight of the lymphoid organs (spleen and thymus) did not showvalues relating to a toxic effect as well (Tab. 1).

                  TABLE 1                                                         ______________________________________                                        Primary antibody production (Jerne test). Evaluation of the                   number of PFC (x ± S.E.) in the spleen of mice immunized                   with SRBC and treated per os with ST 722 at the dose of                       100 mg/kg/day from day -2 through day +2 (immunization                        at day 0).                                                                            Body     Spleen   Thymus                                                      weight   weight   weight PFC/10.sup.6                                                                         PFC/                                  Treatment                                                                             (g)      (mg)     (mg)   cells  spleen                                ______________________________________                                        Control 25.33 ±                                                                             95.50 ±                                                                             64.00 ±                                                                           202 ± 32                                                                          34228 ±                                     0.38     3.19     1.51          5064                                 ST 722  25.12 ±                                                                             94.33 ±                                                                             59.50 ±                                                                           261 ± 34                                                                          44440 ±                                     0.77     4.12     3.62          6087                                 ______________________________________                                    

Test 2

Evaluation of the effect of repeated oral administrations of ST 722 onthe weight of murine lymphoid organs (spleen and thymus).

Experimental Procedure

Male B₆ D₂ F₁ mice (C. River) aged 7 weeks (7-8 animals each group) wereorally treated with the substance ST 722 at the dose of 100 mg/kg/dayfor 7 consecutive days. Twenty-four hours after the last administration,the animals were sacrificed, the organs removed and weighed.

Results

The performed treatment did not provoke any immunotoxic effect on theparameters examined (Table 2).

                  TABLE 2                                                         ______________________________________                                        Weight (x ± S.E.) of murine lymphoid organs after repeated                 treatment oral of the animals with the substance ST 722                       (100 mg/kg/day for 7 consecutive days).                                               Body weight.sup.a                                                                        Spleen weight.sup.a                                                                       Thymus weight.sup.a                            Treatment                                                                             (g)        (mg)        (mg)                                           ______________________________________                                        Control 24.00 ± 0.68                                                                          79.63 ± 3.31                                                                           51.88 ± 2.72                                ST 722  22.74 ± 0.37                                                                          77.88 ± 2.38                                                                           52.88 ± 3.20                                ______________________________________                                         .sup.a mean value (x ± E.S.) of 7-8 samples.                          

Test 3

Evaluation of the effect of repeated oral administrations of ST 722 onthe body, spleen and thymus weight, and on the splenocyte concentrationin mice.

Experimental Procedure

Male B₆ D₂ F₁ mice (C. River) aged 10 weeks (5 animals each group) wereorally treated with the substance ST 722 at the dose of 100 mg/kg/dayfor 5 consecutive days. Twenty-four hours after the last administration,the animals were sacrificed, the organs removed and weighed, and thesplenocyte number determined.

Results

The results, reported in Table 3, showed a lack of specific immunotoxiceffects on the considered parameters following the scheduled ST 722treatment.

                  TABLE 3                                                         ______________________________________                                        Weight of lymphoid organs and spleen concentration after                      repeated oral treatment of mice with the substance ST 722                     (100 mg/kg/day for 5 consecutive days).                                              Body       Spleen     Thymus   Splenoc.                                Treat- weight.sup.a                                                                             weight.sup.a                                                                             weight.sup.a                                                                           number.sup.b                            ment   (g)        (mg)       (mg)     (× 10.sup.-7)                     ______________________________________                                        Control                                                                              27.70 ± 0.48                                                                          75.75 ± 2.36                                                                          41.75 ± 3.59                                                                        7.90                                    ST 722 26.50 ± 0.53                                                                          73.25 ± 3.84                                                                          38.80 ± 3.01                                                                        7.85                                    ______________________________________                                         .sup.a mean value (± S.E.) of 5 animals.                                   .sup.b value from 5 pooled samples.                                      

Test 4

Evaluation of the effect of repeated oral administrations of ST 722 onthe peritoneal macrophage number in mice.

Experimental Procedure

Male B₆ D₂ F₁ mice (C. River) aged 10 weeks (6 animals each group) wereorally treated with the substance ST 722 at the dose of 100 mg/kg/dayfor 5 consecutive days. Twenty-four hours after the last administration,the animals were sacrificed, the peritoneal exudate cells (PEC)collected and the macrophage number determined.

Results

No toxic effect has been observed in PEC macrophage population; on thecontrary, we measured an increase of about 60% in the peritonealmacrophages number of mice treated with the substance ST 722 (Table 4).

                  TABLE 4                                                         ______________________________________                                        Peritoneal macrophage (Mφ) number in mice treated with ST                 722 (100 mg/kg/day for 5 consecutive days).                                                      PEC/              PEC Mφ/                                      Body weight                                                                              mouse.sup.b                                                                             PEC Mφ.sup.b                                                                      mouse.sup.b                              Treatment                                                                             (g).sup.a  (× 10.sup.-6)                                                                     (%)     (× 10.sup.-6)                      ______________________________________                                        Control 28.07 ± 0.63                                                                          2.14      53      1.13                                     ST 722  28.13 ± 1.30                                                                          2.86      63      1.80                                     ______________________________________                                         .sup.a mean value (± S.E.) of 6 animals.                                   .sup.b value from 6 pooled samples.                                      

MICROBIOLOGICAL STUDIES

Test 1

Evaluation of the Minimal Inhibitory Concentration (MIC) in broth of 15new substances for Gram+ bacterial strains.

Experimental Procedure

The following strains were used: Staphylococcus aureus (4);Streptococcus faecalis (8); Bacillus pumilus (1); Bacillus subtilis (1).

The tested substances were: ST 722, ST 982, ST 983, ST 1000, ST 1001, ST1032, ST 1033, ST 1034, ST 1036, ST 1037, ST 1038, ST 1050, ST 1051, ST1052, ST 1053.

MICs were determined by a standard microdilution test, using serialtwo-fold dilutions of the substances in Mueller-Hinton broth.

The inoculum was prepared from an overnight culture in Mueller-Hintonbroth matching the 0.5 Mc Farland (turbidity) standard (BRAY W. E.,Clinical Laboratory Methods, 5th Ed. C. V. MOSBY, St. Louis, Mo. 1957),and was adjusted to a final concentration of 5.0×10⁴ colony-formingunits/ml.

Equal volumes (0.1 ml) of both bacterial suspensions and substancesolutions were distributed in microtiter plates (Falcon, 96 wells, roundbottom), and then placed in a humidified incubator (37° C.) for 18hours.

The results, in terms of MIC values, resistant strain number and meanMIC values versus susceptible strains, are illustrated in Tables 1-6.

In order to establish the type of activity (-static or -cidal) exertedby the substances, the MBC (minimal bactericidal concentration) wasdetermined by subculturing to agar 5 μl from each well where no visiblegrowth had occurred.

Five strains and 10 substances were tested in this way (data not shown),and the absolute coincidence of MIC and MBC values demonstrated that thesubstances have a bactericidal effect.

                  TABLE 1                                                         ______________________________________                                        Minimal Inhibitory Concentration (mcg/ml) of 5 Isovaleryl L-                  Carnitine esters for Gram+ bacterial strains.                                                   ST     ST     ST   ST   ST                                  Bacterial strains 1032   722    1037 1033 1001                                ______________________________________                                        Staphylococcus aureus (303)                                                                     100    6.25   3.12 3.12 6.25                                Staphylococcus aureus (306 MR)                                                                  n.d.   6.25   3.12 3.12 6.25                                Staphylococcus aureus (ATCC                                                                     50     3.12   3.12 3.12 6.25                                14154)                                                                        Staphylococcus aureus (LC1)                                                                     12.5   1.56   3.12 1.56 12.5                                Streptococcus faecalis (501)                                                                    50     3.12   3.12 3.12 3.12                                Streptococcus faecalis (505)                                                                    25     3.12   3.12 1.56 3.12                                Streptococcus faecalis (509)                                                                    n.d.   3.12   1.56 1.56 3.12                                Streptococcus faecalis (516)                                                                    n.d.   1.56   1.56 1.56 3.12                                Streptococcus faecalis (518)                                                                    n.d.   3.12   1.56 1.56 3.12                                Streptococcus faecalis (519)                                                                    n.d.   3.12   1.56 1.56 3.12                                Streptococcus faecalis (R 2)                                                                    n.d.   3.12   1.56 1.56 3.12                                Streptococcus faecalis (UM)                                                                     n.d.   1.56   1.56 1.56 3.12                                Bacillus pumilus (CN 607)                                                                       n.d.   1.56   1.56 1.56 6.25                                Bacillus subtilis (ATCC 6051)                                                                   n.d.   3.12   3.12 3.12 12.5                                ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Mean MIC values (mcg/ml) of 5 Isovaleryl L-Carnitine esters                   for Gram+ bacterial strains.                                                                          Tested  Resistant strains                             Compound                                                                              Mean MIC values*                                                                              strains (MIC > 100)                                   ______________________________________                                        ST 1032 47.50            5      0                                             ST 722  3.12            14      0                                             ST 1037 2.34            14      0                                             ST 1033 2.11            14      0                                             ST 1001 5.35            14      0                                             ______________________________________                                         *Mean MIC values versus susceptible strains.                             

                  TABLE 3                                                         ______________________________________                                        Minimal Inhibitory Concentration (mcg/ml) of 5                                Undecyl L-Carnitine esters for Gram+ bacterial strains.                                      ST     ST     ST    ST    ST                                   Bacterial strains                                                                            1034   1036   1000  982   983                                  ______________________________________                                        Staphylococcus aureus (303)                                                                  3.12   3.12   n.d. >100   >100                                 Staphylococcus aureus                                                                        3.12   3.12   3.12 n.d.   n.d.                                 (306 MR)                                                                      Staphylococcus aureus                                                                        3.12   3.12   1.56 n.d.   n.d.                                 (ATCC 14154)                                                                  Staphylococcus aureus                                                                        1.56   1.56   1.56 3.12   >100                                 (LC1)                                                                         Streptococcus faecalis (501)                                                                 1.56   1.56   n.d. n.d.   n.d.                                 Streptococcus faecalis (505)                                                                 1.56   1.56   n.d. n.d.   n.d.                                 Streptococcus faecalis (509)                                                                 1.56   1.56   n.d. n.d.   n.d.                                 Streptococcus faecalis (516)                                                                 1.56   3.12   n.d. n.d.   n.d.                                 Streptococcus faecalis (518)                                                                 1.56   3.12   3.12 1.56   >100                                 Streptococcus faecalis (519)                                                                 1.56   3.12   3.12 3.12   >100                                 Streptococcus faecalis (R2)                                                                  1.56   3.12   n.d. n.d.   n.d.                                 Streptococcus faecalis (UM)                                                                  1.56   1.56   n.d. n.d.   n.d.                                 Bacillus pumilus (CN 607)                                                                    1.56   1.56   n.d. n.d.   n.d.                                 Bacillus subtilis                                                                            3.12   3.12   n.d. n.d.   n.d.                                 (ATCC 6051)                                                                   ______________________________________                                         n.d. =  not determined.                                                  

                  TABLE 4                                                         ______________________________________                                        Mean MIC values (mcg/ml) of 5 Undecyl L-Carnitine                             esters for Gram+ bacterial strains.                                                                  Tested    Resistant strains                            Compound Mean MIC values*                                                                            strains   (MIC >100)                                   ______________________________________                                        ST 1034  2.00          14        0                                            ST 1036  2.45          14        0                                            ST 1000  2.49          5         0                                            ST 982   2.60          4         1                                            ST 983   >100          4         4                                            ______________________________________                                         *Mean MIC values versus susceptible strains.                             

                  TABLE 5                                                         ______________________________________                                        Minimal Inhibitory Concentration (mcg/ml) of 5 L-Carnitine                    esters for Gram+ bacterial strains.                                                           ST     ST     ST   ST    ST                                   Bacterial strains                                                                             1038   1052   1051 1050  1053                                 ______________________________________                                        Staphylococcus aureus (303)                                                                   6.25   3.12   6.25 6.25  6.25                                 Staphylococcus aureus                                                                         n.d.   3.12   12.5 6.25  6.25                                 (306 MR)                                                                      Staphylococcus aureus                                                                         3.12   n.d.   n.d. n.d.  n.d.                                 (ATCC 14154)                                                                  Staphylococcus aureus (LC1)                                                                   1.56   n.d.   n.d. n.d.  n.d.                                 Streptococcus faecalis (501)                                                                  3.12   3.12   6.25 3.12  6.25                                 Streptococcus faecalis (505)                                                                  3.12   n.d.   n.d. n.d.  n.d.                                 Streptococcus faecalis (509)                                                                  3.12   1.56   6.25 3.12  1.56                                 Streptococcus faecalis (516)                                                                  3.12   n.d.   n.d. n.d.  n.d.                                 Streptococcus faecalis (518)                                                                  3.12   n.d.   n.d. n.d.  n.d.                                 Streptococcus faecalis (519)                                                                  3.12   n.d.   n.d. n.d.  n.d.                                 Streptococcus faecalis (R2)                                                                   3.12   n.d.   n.d. n.d.  n.d.                                 Streptococcus faecalis (UM)                                                                   3.12   n.d.   n.d. n.d.  n.d.                                 Bacillus pumilus (CN 607)                                                                     3.12   1.56   1.56 1.56  1.56                                 Bacillus subtilis                                                                             6.25   3.12   n.d. n.d.  n.d.                                 (ATCC 6051)                                                                   ______________________________________                                         n.d. = not determined.                                                   

                  TABLE 6                                                         ______________________________________                                        Mean MIC values (mcg/ml) of 5 L-Carnitine esters for                          Gram+ bacterial strains.                                                                             Tested    Resistant strains                            Compound Mean MIC values*                                                                            strains   (MIC >100)                                   ______________________________________                                        ST 1038  3.48          13        0                                            ST 1052  2.49          5         0                                            ST 1051  6.56          5         0                                            ST 1050  4.06          5         0                                            ST 1053  4.37          5         0                                            ______________________________________                                         *Mean MIC values versus susceptible strains.                             

Test 2

Evaluation of the Minimal Inhibitory Concentration (MIC) in broth of 16new substances for Gram- bacterial strains.

Experimental Procedure

The following strains were used: Enterobacter (1), Escherichia (3),Klebsiella (3), Proteus (3); Pseudomonas (2); Salmonella (2), Serratia(1).

The tested substances were: ST 712, ST 722, ST 982, ST 983, ST 1000, ST1001, ST 1032, ST 1033, ST 1034, ST 1036, ST 1037, ST 1038, ST 1050, ST1051, ST 1052, ST 1053.

The results, obtained using the same procedure described in Test 1, arereported in Table 7-12.

                  TABLE 7                                                         ______________________________________                                        Minimal Inhibtory concentration (mcg/ml) of 5                                 Isovaleryl L-Carnitine esters for Gram- bacterial strains.                                 ST      ST      ST    ST    ST                                   Bacterial Strains                                                                          1032    722     1037  1033  1001                                 ______________________________________                                        Enterobacter aerogenes                                                                     >100    25        25   100  >100                                 (UM)                                                                          Escherichia coli (76 F)                                                                    n.d.    25      n.d.  n.d.  n.d.                                 Escherichia coli (92 F)                                                                    >100    25      100   >100  >100                                 Escherichia coli                                                                           n.d.    25      n.d.  n.d.  n.d.                                 (R 57B)                                                                       Klebsiella aerogenes                                                                       n.d.    25      n.d.  n.d.  n.d.                                 (SH 1082)                                                                     Klebsiella oxytoca                                                                         n.d.    12.5    n.d.  n.d.  n.d.                                 (552)                                                                         Klebsiella pneumoniae                                                                      n.d.    12.5    n.d.  n.d.  n.d.                                 (7823)                                                                        Proteus mirabilis                                                                          n.d.    >100    n.d.  n.d.  n.d.                                 (32)                                                                          Proteus morganii                                                                           n.d.    100     n.d.  n.d.  n.d.                                 (D.S.)                                                                        Proteus vulgaris                                                                           >100    50      >100  >100  >100                                 (UM)                                                                          Pseudomonas  >100    50      >100  >100  >100                                 aeruginosa (3 E)                                                              Pseudomonas  n.d.    50      n.d.  n.d.  n.d.                                 aeruginosa (10 E)                                                             Salmonella typhi (J)                                                                       n.d.    25      n.d.  n.d.  n.d.                                 Salmonella   >100    25      >100  >100  >100                                 typhimurium (D.S.)                                                            Serratia marcescens                                                                        n.d.    50      n.d.  n.d.  n.d.                                 (A/1)                                                                         ______________________________________                                         n.d. = not determined.                                                   

                  TABLE 8                                                         ______________________________________                                        Mean MIC values (mcg/ml) of 5 Isovaleryl L-Carnitine                          esters for Gram- bacterial strains.                                                                  Tested    Resistant strains                            Compound Mean MIC values*                                                                            strains   (MIC >100)                                   ______________________________________                                        ST 1032  >100          5         5                                            ST 722   35.67         15        1                                            ST 1037  62.50         5         3                                            ST 1033  100           5         4                                            ST 1001  >100          4         5                                            ______________________________________                                         *Mean MIC values versus susceptible strains.                             

                                      TABLE 9                                     __________________________________________________________________________    Minimal Inhibitory Concentration (mcg/ml) of 6                                Undecyl L-Carnitine esters for Gram- bacterial strains.                                      ST   ST   ST   ST  ST  ST                                      Bacterial Strains                                                                            712  1034 1036 1000                                                                              982 983                                     __________________________________________________________________________    Enterobacter aerogenes (UM)                                                                  25   n.d. n.d. n.d.                                                                              n.d.                                                                              n.d.                                    Escherichia coli (76 F)                                                                      25   n.d. n.d. >100                                                                              >100                                                                              >100                                    Escherichia coli (92 F)                                                                      25   25   25   n.d.                                                                              n.d.                                                                              n.d.                                    Escherichia coli (R 57B)                                                                     n.d. 50   >100 n.d.                                                                              n.d.                                                                              n.d.                                    Klebsiella aerogenes (SH 1082)                                                               n.d. 25   100  n.d.                                                                              n.d.                                                                              n.d.                                    Klebsiella oxytoca (552)                                                                     12.5 12.5 50   >100                                                                              >100                                                                              >100                                    Klebsiella pneumoniae (7823)                                                                 12.5 12.5 12.5 n.d.                                                                              n.d.                                                                              n.d.                                    Proteus mirabilis (32)                                                                       >100 >100 >100 n.d.                                                                              n.d.                                                                              n.d.                                    Proteus morganii (D.S.)                                                                      n.d. 100  >100 n.d.                                                                              n.d.                                                                              n.d.                                    Proteus vulgaris (UM)                                                                        100  >100 >100 n.d.                                                                              n.d.                                                                              n.d.                                    Pseudomonas aeruginosa (3 E)                                                                 50   50   100  >100                                                                              >100                                                                              >100                                     Pseudomonas aeruginosa (10 E)                                                               50   50   >100 n.d.                                                                              n.d.                                                                              n.d.                                    Salmonella typhi (J)                                                                         n.d. 25   25   n.d.                                                                              n.d.                                                                              n.d.                                    Salmonella typhimurium (D.S.)                                                                25   100  >100 >100                                                                              >100                                                                              >100                                    Serratia marcescens (A/1)                                                                    50   n.d. n.d. n.d.                                                                              n.d.                                                                              n.d.                                    __________________________________________________________________________     n.d. = not determined.                                                   

                  TABLE 10                                                        ______________________________________                                        Mean MIC values (mcg/ml) of 6 Undecyl L-Carnitine                             esters for Gram- bacterial strains.                                                                  Tested    Resistant strains                            Compound Mean MIC values*                                                                            strains   (MIC >100)                                   ______________________________________                                        ST 712   36.38         15        1                                            ST 1034  45.00         12        2                                            ST 1036  60.71         12        5                                            ST 1000  >100           4        2                                            ST 982   >100           4        4                                            ST 983   >100           4        4                                            ______________________________________                                         *Mean MIC values versus susceptible strains.                             

                  TABLE 11                                                        ______________________________________                                        Minimal Inhibitory Concentration (mcg/ml) of 5 L-                             Carnitine esters for Gram- bacterial strains.                                             ST       ST      ST    ST    ST                                   Bacterial Strains                                                                         1038     1052    1051  1050  1053                                 ______________________________________                                        Enterobacter                                                                              25       >100    >100  >100  >100                                 aerogenes (UM)                                                                Escherichia coli                                                                          n.d.     n.d.    n.d.  n.d.  n.d.                                 (76 F)                                                                        Escherichia coli                                                                          25       >100    >100  >100  >100                                 (92 F)                                                                        Escherichia coli                                                                          100      n.d.    n.d.  n.d.  n.d.                                 (R 57B)                                                                       Klebsiella aerogenes                                                                      50       n.d.    n.d.  n.d.  n.d.                                 (SH 1082)                                                                     Klebsiella oxytoca                                                                        25       >100    >100  >100  >100                                 (552)                                                                         Klebsiella  25       n.d.    n.d.  n.d.  n.d.                                 pneumoniae (7823)                                                             Proteus mirabilis                                                                         >100     n.d.    n.d.  n.d.  n.d.                                 (32)                                                                          Proteus morganii                                                                          100      n.d.    n.d.  n.d.  n.d.                                 (D.S.)                                                                        Proteus vulgaris                                                                          100      >100    >100  >100  >100                                 (UM)                                                                          Pseudomonas 100      >100    >100  >100  >100                                 aeruginosa (3 E)                                                              Pseudomonas 100      n.d.    n.d.  n.d.  n.d.                                 aeruginosa (10 E)                                                             Salmonella typhi (J)                                                                      25       n.d.    n.d.  n.d.  n.d.                                 Salmonella  25       n.d.    n.d.  n.d.  n.d.                                 typhimurium (D.S.)                                                            Serratia    n.d.     n.d.    n.d.  n.d.  n.d.                                 marcescens (A/1)                                                              ______________________________________                                         n.d. = not determined.                                                   

                  TABLE 12                                                        ______________________________________                                        Mean MIC values (mcg/ml) of 5 L-Carnitine esters for                          Gram+ bacterial strains.                                                                             Tested    Resistant strains                            Compound Mean MIC values*                                                                            strains   (MIC >100)                                   ______________________________________                                        ST 1038  41.66         13        1                                            ST 1052  >100          5         5                                            ST 1051  >100          5         5                                            ST 1050  >100          5         5                                            ST 1053  >100          5         5                                            ______________________________________                                         *Mean MIC values versus susceptible strains.                             

Evaluation of the Minimal Inhibitory Concentration (MIC) in Agar of ST722 and ST 712 for 3 Campylobacter and 1 Helicobacter Strains

In this experiment the activity of ST 722 and ST 712 againstCampylobacter and Helicobacter was investigated. To better assess the"in vitro" activity of the compounds mentioned above against thesebacteria, the agar dilution technique was chosen in that it is ratherdifficult ro readily sustain the growth of Campylobacter and notablyHelicobacter in broth cultures.

Bacterial Strains

The strains used were as follows:

Campylobacter fetus ATCC 27374, Campylobacter coli LI 048, Campylobacterjejuni 84-ISS, and Helicobacter pylori NCTC 11637.

The culture medium was Columbia agar base supplemented with 7% (V/V)defribinated horse blood (DHB) for Campylobacter strains, and the samemedium additioned with Vitox (Oxoid) for Helicobacter strain.

The Campylobacter and Helicobacter strains, preserved at -80° C. in DHBcontaining 10% (V/V) glycerol, were thawed and grown, in theirrespective culture medium, in culture flasks, which were incubated for48 hours at 37° C. in a 10% CO₂ atmosphere. After this first growthcycle, the bacteria were allowed to go through a second growth cycle inthe same conditions as above.

The bacterial "patina" was then harvested by resuspension in 4-5 ml ofMueller-Hinton broth, the turbidity of which was then assessed accordingto Mc Farland's criteria (BRAY W. E., Clinical Laboratory Methods, 5thEd. C. V. MOSBY, St. Louis, Mo. 1957).

The Helicobacter suspension was adjusted to an approximate concentrationof 1.0×10⁹ and 1.0×10⁸ organisms/ml, while the Campylobacter suspensionwas adjusted to 1.0×10⁷ and 1.0×10⁶ organisms/ml.

With a Steers-type multipoint inoculator (STEERS E., FOLTZ E. L., GRAVESS., RIDEN J., 1959. Inocula-replicating apparatus for routine testing ofantibacterial susceptibility to antibiotics. Antibiot. Chemoter. 9:307-311) 1-μl volumes of the above standardized suspensions werespot-transferred into the wells of a 96-well microtiter plate, which hadpreviously been filled with 0.2 ml/well of Mueller-Hinton agar 5% DHB,containing the test substances at different concentrations.

The plates was then incubated for 48/72 hours at 37° C. in a 10% CO₂atmosphere.

Results

The MICs for Helicobacter and Campylobacter reported in Table 6 indicatethat both ST 722 and ST 712 possess an antibacterial activity, theformer compound being the most effective one. Besides, the activity ofthe substances is not dependent upon the different infective inocula.

                  TABLE 6                                                         ______________________________________                                        MIC (mcg/ml) in agar of ST 722 and ST 712 for                                 Campylobacter and Helicobacter strains.                                                      ST 722    ST 712                                               Bacterial strains                                                                              I       II      I     II                                     ______________________________________                                        Helicobacter pylori NCTC                                                                       1.56    1.56    3.12  3.12                                   11637                                                                         Campylobacter fetus ATCC                                                                       100     100     100   100                                    27374                                                                         Campylobacter coli LI 048                                                                      3.12    3.12    6.25  6.25                                   Campylobacter jejuni 84-ISS                                                                    12.5    12.5    25    50                                     ______________________________________                                         I = inoculum of 1.0 × 10.sup.5 cells/ml for Helicobacter and            inoculum of 1.0 × 10.sup.3 for Campylobacter.                           II = inoculum of 1.0 × 10.sup.6 cells/ml for Helicobacter and           inoculum of 1.0 × 10.sup.4 for Campylobacter.                      

Evaluation of the "in vitro" Synergistic Effect of ST 722 and BismuthSubcitrate against a Helicobacter pylori Strain

Several studies have shown that Colloidal Bismuth Subcitrate (CBS)precipitates in the presence of the low pH of the gastric juice, andforms a complex with proteins on the ulcer base which may create aprotective layer against agressive factors, thereby allowing healing ofthe ulcer lesion to occur (WAGSTAFF, A. J., BENFIELD, P. and MONK J. P.,1988. Colloidal Bismuth Subcitrate. A rewiew of its Pharmacodynamic andPharmacokinetic Properties, and its Therapeutic use in Peptic UlcerDisease. Drugs 36, 132-157).

These observations together with the recent demonstration of anantibacterial activity of CBS versus Helicobacter (MIC values rangingfrom 4 to 32 mcg/ml) (Mc NULTY, C. A. M., DENT, J. and WISE, R., 1985.Susceptibility of clinical isolates of "Campylobacter pyloridis" to 11antimicrobial agents. Antimicrob. Agents Chemoter. 28, 6, 837-838) haveled several investigators to study the possible synergistic effect ofCBS and antibiotics endowed with anti-Helicobacter activity aimed atobtaining a complete and persistent eradication of the bacteria from thestomach (VAN CAEKENBERGHE, D. L. and BREYSSENS, J., 1987. In vitrosynergistic activity between Bismute Subcitrate and variousantimicrobial agents against "Campylobacter pyloridis" ("C. pylori").Antimicrob. Agents Chemoter. 31, 9, 1429-1430).

In the light of these findings, we looked for a possible synergisticeffect of CBS and ST 722 against a strain of Helicobacter pylori.

MATERIALS AND METHODS

Substances

CBS from the commercially available product De-Nol® (Gist-Brocades,Netherland), i.e. ammonium and potassium double salt of colloidalbismuth citrate hydroxide, was utilized by preparing a suspension of 10mg/ml CBS in 1N NaOH. After diluting 1:10 in Mueller-Hinton broth, 8serial two-fold dlitutions in the same medium were made. The compound ST722 was dissolved in Mueller-Hinton broth at a starting concentration of100 mcg/ml, then 7 serial two-fold dilutions were carried out.

Bacterial Strain

The "in vitro" anti-Helicobacter activity of both CBS and ST 722, aloneand in association, was tested by utilizing the strain H. pylori NCTC11637.

Inoculum Preparation

A 24-hour culture of Helicobacter in Columbia agar medium supplementedwith Vitox (Oxoid) and 7% defribinated horse blood is washed withMueller-Hinton broth.

Following densitometric evaluation (reading at 600 nm), the bacterialsuspension was adjusted to a concentration of 1.0×10⁹ cells/ml and0.2-ml volumes were distributed into each well of a 96-well U-bottomedmicrotiter plate.

A 1-μl volume of this bacterial suspension was then distributed, bymeans of a Steers-type replicator apparatus, into the wells ofmicrotiter plates containing the substances, either alone or inassociation. The final bacterial inoculum contained approximately1.0×10⁶ bacterial cells.

Experimental Procedure

The procedure described by Garrod et al. (GARROD, L. P. and WATERWORTH,P. M., 1962. Methods of testing combined antibiotic bactericidal actionand the significance of the results. J. Clin. Pathol. 15, 328-338) wasutilized.

Briefly, 250-μl volumes of each test substance, either alone or admixedin all the possible combinations, were additioned to 4.5 ml ofMueller-Hinton agar containing 5% defribinated horse serum. Volumes of0.2 ml/well of the resulting solution were distributed into the wells ofa microtiter plate, then seeded with 1 μl/well of a standardizedbacterial suspension, and finally incubated for 48 hours at 37° C. in a10% CO₂ atmosphere.

Criteria for the Evaluation of the Results

In order to evaluate the results obtained with combinations ofantibiotics it was utilized the method (KROGSTAD, D. J. and MOELLERING,R. C., 1983. Antimicrobial combinations. In LORIAN, V., Antibiotics inLaboratory Medicine, 15, 537-549) based on the calculation of theFractional Inhibitory Concentration Index (FIC index).

FIC index is calculated as follows: ##EQU1## Where (A) is the MIC ofsubstance A in the association and (MIC_(A)) is the MAC of the organismto substance A alone. FIC_(A) is the fractional inhibitory concentrationof substance A. (B), (MIC_(B)) and FIC_(B) are defined in the samefashion for substance B.

Based on these criteria, the effect of a combination of 2 substances isreferred to as 'synergistic" when the FIC index is ≦0.5, "additive" witha FIC index=1, and "antagonist" when FIC index is >1.

Finally, an "indifferent"effect occurs when the result with 2 substancesis not significantly different from the result with the most effectivesubstance alone, i.e. when FIC index=FIC_(A) or FIC_(B).

Results

The data reported in Table 6 show that the MIC of ST 722 and BismuthSubcitrate against H. pylori is 1.56 mcg/ml and 6.25 mcg/ml,respectively.

The combination of the above substances results to be inhibitory at 0.19mcg/ml ST 722 and 3.12 mcg/ml CBS.

The relevant FIC index is equal to 0.62. indicating that the associationis not merely additive although not overly synergistic.

                  TABLE 6                                                         ______________________________________                                        Evaluation of the "in vitro" synergistic effect of ST 722                     and Colloidal Bismuth Subcitrate (CBS) against                                Helicobacter pylori.                                                          Substances  MIC.sup.a    FIC.sup.b                                                                            FIC index.sup.c                               ______________________________________                                        ST 722      1.56         0.12                                                 CBS         6.25         0.50                                                 ST 722 + CBS                                                                              0.19 + 3.12         0.62                                          ______________________________________                                         .sup.a minimal inhibitroy concentration (mcg/ml).                             .sup.b fractional inhibitory concentration                                    .sup.c fractional inhibitory concentration index.                        

Evaluation of the Bactericidal Effect of ST 722 against Helicobacterpylori (Planktonic and Sessile Bacteria) and Evaluation of the CytotoxicEffect of ST 722 Versus Unifected or H. pylori Infected HEp-2 Cells

The investigation aimed at evaluating the difference in susceptibilityto ST 722 of planktonic and sessile Helicobacter cells, using in thelatter case an "in vitro" experimental model, which closely resembledthe clinical condition occurring "in vivo" in type-B gastritis. We usedHEp-2 cells, an epithelial cell line which harbors the same membraneglycolipid receptors for H. pylori as those harbored by the mucous cellsof the antral gastric mucosa (MEGRAUD, F., TRIMOULET, P., LAMOULIATTE,H. and BOYANOVA, L., 1991. Bacterial effect of Amoxicillin on "H.pylori" in an in vitro model using epithelial cells. Antimicrob. AgentsChemoter. 35 (5): 869-872 and LINGWOOD, C. A., LAW, H. and PELLIZANI,A., 1989. Gastric glycerolipid as a receptor for Campylobacter pylori.Lancet i: 238-241). Additionally, the viability of HEp-2 cells (eitherinfected or not) was evaluated following "in vitro" ST 722 treatment.The degree of cytotoxicity was assessed by cytofiuorimetric analysis ofPropidium Iodine-stained HEp-2 cells.

MATERIALS AND METHODS

Cell Line

An HEp-2 epithelial cell line (a human larynx carcinoma) was used topermit the attachment of Helicobacter cells (sessile cells).

Bacterial Strain

Helicobacter pylori NCTC 11637.

HEp-2 Culture Medium

DMEM supplemented with 10% FCS.

Substance

The antibacterial and cytotoxic activity of ST 722 against Helicobacterand HEp-2 cells, respectively, was evaluated by using 3 different ST 722concentrations, namely 0.1, 1.56 (i.e. the MIC value vs. H. pylori NCTC11637) and 4 mcg/ml.

1. Experimental Procedure for the Evaluation of the Anti-HelicobacterActivity

A confluent HEp-2 cell culture is prepared, which is obtained afterincubating the cell line in tissue culture flasks for 24 hours (37° C.,10% CO₂, and ˜96% humidity) in Columbia agar medium supplemented withVitox and 7% defribinated horse serum. The bacteria are first harvestedwith DMEM medium 10% FCS, and then inoculated onto the HEp-2 cells toobtain a concentration of 5.0×10⁶ CFU per flask (i.e. 2.0×10⁵ CFU/cm²).

Bacterial adhesion to the epitelial cells is favored by incubation for 2hours at 37° C. and 5% CO₂. After washing to remove the non attachedbacteria. ST 722 is added (5 ml of 3 different concentrations) to theHEp-2 cell culture flasks.

Two additional flasks are added with 5 ml medium lacking ST 722, asHelicobacter growth controls.

At a predetermined time, the flasks were taken, the medium wasdiscarded, and the cells were washed. The cells were harvested with arubber policeman in 1 ml of phosphate-buffered saline and disrupted withan Ultraturax homogenizer.

After making geometric dilutions of the suspensions (0.1 ml of eachdilution inoculated onto each plate in triplicate), the plates wereincubated for 7 days in a microaerobic atmosphere, and the plates with30 to 200 CFU were counted.

The ST 722 activity against Helicobacter planktonic cells was determinedin the same experimental conditions in the absence of HEp-2 cells.Finally, the results are expressed as CFU per flask of each sample withrespect to the relevant control (i.e. sessile or planktonic cultures).

2. Experimental Procedure for the Evaluation of the HEp-2 Cell Viability

The HEp-2 cells were seeded in 24-well plate at a concentration of5.0×10⁴ cells/well and incubated for 72 hours at 37° C. in a 5% CO₂atmosphere to yield a confluent monolayer. After washing with Hank's(HBSS) solution, 1 ml of H. pylori suspension (4.0×10⁵ cells/ml) wasadded into each well.

After incubating 2 hours at 37° C., to allow the bacteria to attach tothe HEp-2 cell monolayer, the plates were washed once with HBSS, and ST722 (at 3 different concentrations in DMEM 10% FCS) was finally added.

Three and twenty-four hours following ST 722 addition, the cells weretrypsinized and harvested by centrifugation at 800 g, The cell pelletswere then resuspended in 500 μl PBS and additioned with 6 μl of a 1mcg/ml Propidium Iodine stock solution in PBS, The same procedure wasfollowed for the control, i.e. HEp-2 cells not infected with H. pylori.The cell suspensions were finally analyzed by means of a FACScancytofluorimeter.

Results

1. The Helicobacter strain utilized in this "in vitro" experimentalmodel turned out to loosely attach to HEp-2 cells. In fact, startingfrom an inoculum of 1.0×10⁶ cells only 9.5×10² cells did adhere to theepithelial cells after 3-hour incubation, increasing up to 4.2×10⁵following 24-hour incubation (Table 7). The number of sessile bacterialcells was reduced (approximately 50%) after a 3-h contact with 4 mcg/mlST 722, while only 1.56 mcg/ml ST 722 was necessary to attain the sameresult after a 24-h contact. Similarly, ST 722 was more active againstplanktonic cells after incubation for 24 hours, being able to reduce thenumber of Helicobacter cells from 5.35×10⁸ down to 1.85×10⁶ at aconcentration of 4 mcg/ml.

However, it has to be underlined that although ST 722 appeared to bemore effective against planktonic cells, a complete medium sterilizationwas not attained.

It is likely that the adopted experimental conditions negativelyinterfered in obtaining the same result found in the experiment carriedout in agar medium (MIC=1.56 mcg/ml).

2. The selective staining of dead HEp-2 cells, detected bycytofluorimetric analysis of Propidium Iodine-stained cells, allowed toascertain that ST 722 is devoid of any cytotoxicity versus HEp-2 cells.

The results indicate that even an ST 722 concentration of 4 mcg/ml,which was effective against Helicobacter sessile cells, was unable toinduce detectable cytotoxicity versus the HEp-2 epithelial cells (datanot shown).

                  TABLE 7                                                         ______________________________________                                        Bactericidal effect of ST 722 against H. pylori planktonic                    and sessile cells. The results are expressed as CFU per flask.                Experimental Sessile cells                                                                             Planktonic cells                                     samples      (CFU per flask)                                                                           (CFU per flask)                                      ______________________________________                                        3-h contact                                                                   Control      9.50 × 10.sup.2                                                                     9.00 × 10.sup.5                                0.1 mcg/ml   9.50 × 10.sup.2                                                                     9.50 × 10.sup.5                                1.56 mcg/ml  9.50 × 10.sup.2                                                                     3.50 × 10.sup.5                                4.0 mcg/ml   4.50 × 10.sup.2                                                                     1.00 × 10.sup.5                                24-h contact                                                                  Control      4.25 × 10.sup.5                                                                     5.35 × 10.sup.8                                0.1 mcg/ml   4.35 × 10.sup.5                                                                     9.00 × 10.sup.7                                1.56 mcg/ml  1.80 × 10.sup.5                                                                     4.05 × 10.sup.6                                4.0 mcg/ml   1.75 × 10.sup.5                                                                     1.85 × 10.sup.6                                ______________________________________                                    

Evaluation of the Protective Effect of ST 722 in a SubcutaneousExperimental Infection with Staphylococcus aureus in Mice

MATERIALS AND METHODS

Animals

Male CD₁ (C. River) mice aged 9 weeks were used (4 animals per group).

Bacterial Strain

A pathogenic strain of Staphylococcus aureus (S. aureus LC₁), isolatedfrom a systemic infection in nude mice, was utilized.

This strain possesses a virulence such that the DL₅₀ is equal to 7.3×10⁶cells/mouse, when i.p. inoculated in 5% gastric mucin. ST 722 exibited aMIC of 1.56 mcg/ml for the strain of Staphylococcus used.

Inoculum Preparation

The bacterial cells (kept under liquid nitrogen) are thawed and seededin 10 ml of TSB medium and finally incubated for approximately 18 hoursat 37° C.

The culture is then diluted in sterile saline so as to have 2.2×10⁷bacterial cells in a volume of 0.2 ml.

Treatment

Protocol 1

ST 722 was administered subcutaneously, immediately after bacterialinoculation, at the doses of 5, 20, and 50 mcg in 0.2 ml of sterilesaline (single treatment).

Protocol 2

ST 722 was administered subcutaneously at a dose of 50 mcg (in 0.2 ml ofsterile saline) immediately after bacterial inoculation and again 5hours later, i.e. 100 total mcg per mouse (double treatment).

Experimental procedure

The "in vivo" experimental model described by Grunberg et al. (GRUNBERG,E., BERGER, J., BESKID, G., CLEELAND, R., PRINCE, N. H. and TITSWORTH,E., 1967. Studies on the "in vitro" and "in vivo" chemotherapeuticproperties of the antibiotics myxin. Chemotherapia 12, 272-281) wasutilized.

Briefly, it consisted of a subcutaneous injection into the center of theabdominal wall of the standardized bacterial suspension followed by thes.c. administration of the substance in the same area according to the 2protocols above.

After 24 hours, the animals were sacrificed, and the abdominal wall(including the peritoneum) was exicised from each animal.

The tissue samples were first homogenized by a Potter-Elvehjem tissuegrinder in 5 ml of a sterile saline, then a bacterial count wasperformed by plating the samples onto a S. aureus selective medium(Baird-Parker agar additioned with Ey tellurite enrichment), whichallows to easily detect the number of Staphylococcus colonies.

Evaluation of the Results

The number of colonies scored in a series of dilutions for each singlesample consents to calculate the number of bacterial cells present inthe infected tissue sample as follows: ##EQU2## Where Z_(i) is thenumber of dilutions performed, N_(i) is the number of plates preparedfor each dilution, and C_(i) is the total number of bacteria scored ineach dilution.

Results

The subcutaneous infection induced in mice represents a particular modelof topical infection, which is utilized to test the protective effect ofa subcutaneous treatment with a substance, i.e. ST 722, whosepenetration ability after simple local deposition on epidermis is notyet fully established.

Protocol 1

A dose of 5 mcg of ST 722 was unable to reduce the infective process,while doses of 20 e 50 mcg dose-dependently reduced in an appreciablemanner the bacterial infection extent (Table 8), althought did notcompletely eradicate the bacteria from the tissue samples examined 24hours following the bacterial inoculation.

Protocol 2

The same type of results were obtained after a double administration ofST 722 (50 mcg, twice). In this case, in fact, the treatment causes areduction in the number of bacterial cells from 5.7×10⁷ down to 3.1×10⁵(Table 13).

                  TABLE 8                                                         ______________________________________                                        Protective effect of ST 722 (single treatment) in a                           subcutaneous infective model with S. aureus in mice.                          Results are expressed as number of bacteria/mouse                             (mean values from 4 animals).                                                 Treatment    Number of bacteria/mouse                                         ______________________________________                                        Control      1.11 × .sup.10.sup.8                                       ST 722  5 mcg                                                                              1.21 × 10.sup.8                                            ST 722 20 mcg                                                                              1.21 × 10.sup.8                                            ST 722 50 mcg                                                                              5.61 × 10.sup.5                                            ______________________________________                                    

                  TABLE 9                                                         ______________________________________                                        Protective effect of ST 722 (double treatment) in a                           subcutaneous infective model with S. aureus in mice.                          Results are expressed as number of bacteria/mouse                             (mean values from 4 animals).                                                 Treatment   Number of bacteria/mouse                                          ______________________________________                                        Control     1.11 × .sup.10.sup.8                                        ST 722      5.61 × 10.sup.5                                             ______________________________________                                    

Evaluation of the Antibacterial Activity of the Undecyl Alcohol

To ascertain whether the antimicrobial activity was due to the undecylalcohol (a possible product from the hydrolysis of the undecyl esters,such as e.g. ST 722 and ST 712), different "in vitro" tests wereperformed to overcome the difficulties stemming from the insolubility ofthe undecyl alcohol in the assay medium.

To circumvent this problem, three different experimental approaches werechosen obtaining the following results:

1) Undecyl alcohol emulsified in Tween 80 and tested up to theconcentration of 300 mcg/ml in agar seeded with 7 Gram- bacterialstrains (Agar dilution test).

Results: no activity.

2) Undecyl alcohol absolute (0.05 ml) in agar seeded with 4 Gram-bacterial strains (Agar diffusion test).

Results: no activity.

3) Undecyl alcohol emulsified in Tween 80 and tested up to theconcentration of 400 mcg/ml in broth seeded with 4 Gram- bacterialstrains and 1 Gram+ bacterial strain (Broth dilution test).

Results: no activity.

We claim:
 1. An ester of acyl L-carnitine of the general formula##STR6## wherein: R is a straight or branched saturated aliphatichydrocarbon carboxylic acyl group having 2-16 carbon atoms,n is aninteger from 7 to 15, and X⁻ is the anion of a pharmacologicallyacceptable acid.
 2. An ester of claim 1 wherein R is isobutyryl orisovaleryl.
 3. An ester of claim 1 wherein n is
 10. 4. An ester of claim1 wherein X⁻ is selected from: chloride; bromide; iodide; acidaspartate; acid citrate; tartrate; acid phosphate; acid fumarate;glycerophosphate; glucosephosphate; lactate; acid maleate; orotate; acidoxalate; acid sulphate; trichloroacetate, trifluoroacetate andmethansulphonate.
 5. Isovaleryl L-carnitine undecyl ester chloride. 6.Isobutyryl L-carnitine undecyl ester chloride.
 7. An orally orparenterally administrable antibacterial pharmaceutical compositioncomprising an ester of claims 1, 4, 5, or 6 as active ingredient inantibacterially effective amount and a pharmacologically acceptableexcipient.
 8. The antibacterial pharmaceutical composition of claim 7for treating diseases the aetiologic agent whereof is a bacterium of thegenus Campylobacter and the active ingredient is present in amountantibacterially effective against said bacterium.
 9. The pharmaceuticalcomposition of claim 7 for treating an infection sustained by Gramnegative or Gram positive bacteria wherein the active ingredient ispresent in an antibacterially effective amount against said bacteria.10. An ester of claim 4, wherein R is isobutyryl or isovaleryl.
 11. Anester of claim 4, wherein n is
 10. 12. The antibacterial pharmaceuticalcomposition of claim 7 for treating diseases the aetiologic agentwhereof is a bacterium of the genus Helicobacter and the activeingredient is present in amount antibacterially effective against saidbacterium.